Capturing bacteria by magnetic separation is a well-established technique that allows the collection of selectively concentrated pathogens for further analysis and identification. Immunomagnetic separation (IMS) technology is able to isolate bacteria strains possessing specific and characteristic surface antigens [Clinical Microbiology Reviews, 7(1), 43-54, 1994; Sensors, 9, 717-730, 2001; Critical Reviews in Microbiology, 30(1), 7-24, 2004]. Further identification of the concentrated bacteria is performed by traditional biochemical, immunologic, or molecular methods. Such technology can be used in combination with polymerase chain reaction (PCR) techniques to improve sensitivity and decrease detection time [FEMS Microbiology Letters, 176(2), 285-289, 1999; Int J Food Microbiol., 99(1), 47-57, 2005, PLoS ONE, 8(12), e82376, 2103]. Highly sensitive bacteria quantification is achieved by indirect electrochemistry detection and immunomagnetic separation with formation of a sandwich complex [Sensor, 15, 12034-1205, 2015]. Specific and sensitive detection of bacteria has been recently demonstrated using aptamer-coated magnetic beads and antibiotic-capped gold nanoclusters [Anal. Chem., 88 (1), 820-825, 2016].
Fast detection is also obtained by using magnetic beads in combination with lyotropic liquid crystals [U.S. Pat. No. 6,171,802 B1, U.S. Pat. No. 7,745,220 B2, U.S. Pat. No. 6,411,354 B1, U.S. Pat. No. 6,570,632 B2]. The formation of an immune complex by binding of antibody coated magnetic microbeads to bacteria creates a deformation in an aligned liquid crystal (LC), whereby a detectable optical signal is generated. Lyotropic liquid crystals of a non-surfactant nature which are known as lyotropic chromonic liquid crystals are best suited for such technology, due their non-toxic nature, and therefore compatibility with biological systems. Similarly to surfactant based lyotropic liquid crystals, chromonics form a liquid crystal phase when mixed with a solvent, generally water or physiological buffers. The mechanism of aggregation does not involve the formation of micelles at a critical concentration, but the chromonic molecules stack face to face, forming polydisperse, rod-like aggregates [J. Lydon, Chromonics, in: Handbook of Liquid Crystals (Wiley-VCH, Weinheim, 1998) v. 2B, p. 981 and Current Opin. Col. Inter. Sci. 3, 458 (1998)]. The aggregation is driven by weak non covalent interactions such as π-π attraction, and the length of the aggregates depends on concentration and temperature.
Detection of pathogenic material/bacteria using chromonics requires the material to align in a specific direction (uniform planar or homeotropic) when confined in a closed cell. The alignment can be obtained using aligning materials such as polyimides. Chromonic azodyes can be readily aligned homeotropically on hydrophobic substrates with very low surface tension as disclosed in Applicant's currently pending patent application for System and Method for Detecting Pathogens on Treated and Untreated Substrates Using Liquid Crystal Chromonic Azo Dye, U.S. Patent Application Publication No. US2016/0139054 A1, published on May 19, 2016.
An exemplary prior art detection technology having a non-surfactant lyotropic chromonic liquid crystal (LCLC) cell is designated generally by the numeral 40 in FIG. 1. The cell 40 includes a pair of opposed substrates or boundary plates 42, which are sealed in a manner known in the art and which contains lyotropic liquid crystal material 44. Qualitatively, the difference between LCLC's and surfactant type lyotropic materials is that LCLC molecules, designated generally by the numeral 46 in FIG. 1, are disc-like or plank-like rather than rod-like. The polar hydrophilic parts 48 form the periphery of each molecule, while the central core 50 is relatively hydrophobic. This distinction creates a range of different ordered structures. Individual disc-like molecules may form cylindrical aggregates 54 in water 56. The direction of average molecular orientation is defined by the orientation of the normals to the planes of the plank-like or disc-like molecule and indicated by, a director 60 disposed along the longitudinal axis n of the cylindrical aggregate showing the direction of orientation. In the example depicted in FIG. 1, the long axes of the aggregates are oriented in a direction parallel to the bounding plates or substrates. However, the aggregates do not necessarily align in the same parallel direction. Such an alignment requires a special treatment of the substrates. An efficient detection of ligands is possible when a ligand-receptor complex disturbs a uniform alignment of the liquid crystal in an LC cell.
Another exemplary prior art non-surfactant lyotropic liquid crystal cell with homeotropic alignment used for the detection and amplification of ligands is shown schematically in FIG. 2 and designated generally by the numeral 70. The cell 70 includes a pair of opposed substrates or boundary plates 72, which are sealed in a well-known manner. The cell contains a lyotropic chromonic liquid crystal material 74 in water 76. The difference between material 74 and the prior art chromonic material 44 of FIG. 1 lies in the preferential alignment of chromonic material 74. On a variety of substrates, this alignment is homeotropic, meaning the long axes of the aggregates (one of which is shown at n) are oriented in a direction perpendicular to the bounding plates. Director 78 shows the direction of orientation. An efficient detection of ligands is possible when the ligand-receptor complex disturbs the homeotropic alignment of the liquid crystal in the liquid crystal cell.
The efficiency of this type of detection depends not only on the formation of an immune complex by ligand-receptor binding, but requires the formation of ligand-receptor clusters large enough to create a deformation of the aligned LC creating an optical signal intensity which is higher than the background signal intensity generated by the receptor-receptor pairs' spontaneous aggregation.
To be able to improve the technology further by eliminating the background signal and increasing detection sensitivity a need exists for an alternative simplified capture and amplification mechanism which maximizes the effect of ligand-receptor pair by creating a deformation in the aligned LC and minimizing the effect of spontaneously forming receptor-receptor pairs.